This project focuses on identification and functional characterization of human 3p recessive oncogenes (tumor suppressor genes, TSGs), with the initial emphasis being on discovery of TSGs in a 630 kb, completely sequenced, 3p21.3 region found to be homozygously deleted in lung and breast cancers. Our overall hypothesis is that biallelic inactivation or haploinsufficiency of one or more of these genes is of fundamental importance in the pathogenesis of many human cancers and that this inactivation occurs early in the multistep process of carcinogenesis. We have just completed the isolation and initial characterization of 25 genes in this area (23 of which are new) and found two, RASSF1A (Ras interacting protein with a DAG binding domain), and SEMA3B (secreted class III semaphorin) to have biallelic expression loss and potent tumor suppressing activity. We also found several others to have some characteristics of TSGs (loss of expression, mutation, suppression of the malignant phenotype) including CACNA2D2, 101F6, NPR21/G21, BLU, FUSI, HYAL1, and FUS2. The current proposal will: Confirm the tumor suppressing function of RASSF1A and SEMA3B and further implicate or dismiss the other candidate 3p21.3 TSGs in multiple human cancer cell lines with different genetic abnormalities by in vitro and in vivo xenograft assays (Aim 1); Confirm that tumor acquired loss of expression of RASSF1A and SEMA3B and some of the other 3p21.3 TSG candidates occurs in human cancers but not in normal tissues, and is explained by tumor acquired methylation of CpG islands in their promoter regions (Aim 2); Study the potential mechanisms for tumor suppression of RASSF1A and SEMA3B using human cancer cell lines and thus determine the pathways involved in RASSF1A and SEMA3B suppression of the tumor phenotype; in the process also determine if acquired and germline amino acid sequence alterations in these genes inactivate their function potentially predisposing to cancer development (Aim 3); Determine whether a Sema3b knockout mouse model challenged with carcinogens confirms SEMA3B as a TSG and also test whether SEMA3B haploinsufficiency is sufficient for aiding carcinogen induced lung cancer in the mouse. (Aim 4). The characterization of these gene(s) should ultimately have translational benefit for the development of new cancer diagnostics and therapeutics, including use in cancer early detection, prevention, and treatment.